1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

Carcinogenicity

Description of key information

Melamine cyanurate is non genotoxic. Experimental data on carcinogenicity is not available.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

1978 to 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP studies are considered as top quality studies, even if GLP was formally not stated. The studies are peer reviewed. The actual study is assigned as valid without restriction. A reliability of 2 must be assigned for read-across.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: NTP standards

Deviations:

not specified

Principles of method if other than guideline:

A carcinogenesis bioassay of melamine was conducted by feeding diets containing melamine in two concentrations to groups of rats of each sex for 103 weeks. Groups of rats served as controls.

GLP compliance:

not specified

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: 4 weeks.
- Housing: Rats were housed five per cage in polycarbonate cages covered with nonwoven polyester filter sheets. Racks and filters were changed once every 2 weeks. Cages, bedding, and glass water bottles (equipped with stainless steel sipper tubes) were replaced twice per week.
- Diet: Purina Laboratory Chow. Ralston Purina Co. Stainless steel feed containers were changed once per week.
- Water: Tap water (acidified with hydrochloric acid to pH 2.5).
Test diets, control diets, and tap water were available ad libitum.
- Bedding: Absorb-Dri hardwood chips.
- Acclimation: 2 weeks
- Randomization: Animals were assigned to individual cages according to a table of random numbers. The cages were then distributed to control and dosed groups according to another table of random numbers.
ENVIRONMENTAL CONDITIONS
- Temperature, humidity: The temperature in the animal rooms was 22 °- 26 °C and the relative humidity was 30 %-70 %.
- Air changes (per hr): Room air was changed 12-15 times per hour. The air was prefiltered with DRICO air filters and then filtered with HEP A filters.
- Photoperiod: Fluorescent lighting provided illumination 12 hours per day.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with: Test diets were prepared by first mixing a small amount of Purina B Lab Chow and the required amount of melamine with a mortar and pestle and then adding this premix to the required amount of animal meal and mixing for 10 to 30 minutes in a Patterson-Kelly@ twinshell blender equipped with an intensifier bar.
- Storage temperature of food: Test diets were stored in the freezer for no longer than 3 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Prepared diets containing 100 000 ppm melamine were analyzed and were found to be stable for 2 weeks at temperatures up to 45 °C.
Control animals were fed Purina Lab Chow.
Dosed feed samples from the chronic studies were analyzed periodically by ultraviolet spectroscopy. The results obtained indicate that only one of the formulations analyzed was slightly (+ 10.6 %) out of specifications (>± 10 %).
Results from three separate referee analyses verified the accuracy of the formulations.

Duration of treatment / exposure:

103 weeks.

Frequency of treatment:

/

Post exposure period:

No.

Remarks:

Doses / Concentrations:
Diets containing 0 - 4 500 - 9 000 ppm melamine were fed to groups of 50 female rats.
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
Diets containing 0 - 2 250 - 4 500 ppm melamine were fed to groups of 50 male rats.
Basis:
nominal conc.

No. of animals per sex per dose:

50 female and 50 male rats per dose group and control group. One control male rat was discarded at week 13, when a sexing error was discovered.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Doses were selected according to 28-days and 13-weeks studies.

Positive control:

Not required.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for morbidity or mortality.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were recorded monthly.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights by cage were recorded once per week for the first 13 weeks, monthly until week 91, and then every 2 weeks. The mean body weight of each group was calculated by dividing the total weight of all surviving animals in the group by the number of surviving animals in the group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption by cage was recorded once per week for the first 13 weeks, monthly until week 91, and then every 2 weeks. The average feed consumption per animal was calculated by dividing the total feed consumption measured for all cages in a group by the number of surviving animals in the group.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No.
OPHTHALMOSCOPIC EXAMINATION: No.
HAEMATOLOGY: No.
CLINICAL CHEMISTRY: No.
URINALYSIS: No.
NEUROBEHAVIOURAL EXAMINATION: No.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Moribund animals and animals that survived to the end of the bioassay were killed with carbon dioxide and immediately necropsied.
- Examinations for grossly visible lesions were performed on major tissues or organs.
- Tissues were preserved in 10 % neutral buffered formalin embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
- The following were examined microscopically: gross lesions, skin with mammary gland, mandibular lymph node, salivary gland, sternum with bone marrow, larynx or anterior trachea, esophagus, thyroid, parathyroid, lungs with mainstem bronchi, heart, stomach (glandular and nonglandular), duodenum, large intestine, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenal glands, urinary bladder, entire gonads, prostate or uterus, brain, and pituitary gland.
- Necropsies were performed on all animals found dead and on those killed at the end of the study, unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on study in each group.
- The pathology report and selected slides were evaluated by the NTP Pathology Working Group. The classification of neoplastic nodules was done according to the recommendations of Squire and Levitt (1975) and the National Academy of Sciences (1980). The diagnoses represent a consensus of contracting pathologists and the NTP Pathology Working Group.

Statistics:

Data on this experiment were recorded in the Carcinogenesis Bioassay Data System. The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, body weight, and individual pathologic results, as recommended by the International Union Against Cancer.
Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958) and are presented in the form of graphs. Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extensions of Cox's methods for testing for a dose-related trend. All reported P values for the survival analyses are two-sided.
The incidence of neoplastic or nonneoplastic lesions has been given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators included only those animals for which that site was examined histologically.
However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors) prior to histologic sampling, or when lesions could have appeared at multiple sites (e.g., lymphomas), the denominators consist of the numbers of animals necropsied.
For the continued description of the statistical methods, see under "Any other information on material and methods" (due to maximum number of letters available in this field).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS: No compound-related clinical signs were observed.
BODY WEIGHT AND WEIGHT GAIN: After week 20, mean body weights of dosed rats of each sex were lower than those of the controls.
FOOD CONSUMPTION: The average daily feed consumption per rat by low- and high-dose rats was 97 % and 99 % that of the controls for males and 99 % and 99 % for females.
SURVIVAL: Estimates of the probabilities of survival of male and female rats fed diets containing melamine at the concentrations of this bioassay, together with those of the control group, are shown by the Kaplan and Meier curves, see also the attached Figures. One control rat in the male group was mis-sexed and removed from the study. The survival of the high-dose group of male rats was significantly reduced when compared with that of the controls (P=0.03). No significant differences were observed between any other groups of either sex.
In male rats, 30/49 (61 %) of the controls, 30/50 (60 %) of the low-dose, and 19/50 (38 %) of the high-dose group lived to the termination period of the study at 105 weeks; 30/50 (60%) of the high-dose group of male rats survived 92 weeks of the study. In female rats, 34/50 (68 %) of the controls, 30/50 (60 %) of the low-dose, and 27/50 (54 %) of the high-dose group lived to the termination period of the study at 105 weeks. The survival data include one low-dose and one high-dose male rat that died during the termination period of the study. For statistical purposes, these animals are considered to have been killed at the end of the study.

PATHOLOGY:
Histopathologic findings on neoplasms in rats and findings on nonneoplastic lesions are summarized in the Tables in the attachment.
Incidences of urinary bladder and kidney lesions are presented also in Tables there. Other Tables contain the statistical analyses of those primary tumors that occurred with an incidence of at least 5 % in one of the three groups.

Urinary Bladder:
Transitional-cell carcinomas in the urinary bladder of male rats occurred with a statistically significant (P = 0.002) positive trend (controls, 0/45; low-dose, 0/50; high-dose, 8/49, 16 %) and the incidence in the high-dose group was significantly higher (P = 0 .016) than that in the controls.
The combined incidence of transitional cell carcinomas and papillomas showed a statistically significant (P < 0.001) positive trend (controls, 0/45; low-dose, 0/50; high-dose, 9/49, 18 %) and the incidence in high-dose rats was significantly higher (P = 0.008) than that in the controls.
These tumors were not observed in statistically significant proportions in female rats (0/49, 1/49, 1/47).

The transitional-cell carcinomas were visualized grossly as 1- to 1.5-cm masses attached to the mucosal surface of the urinary bladder. Seven of the eight high-dose male rats with transitional-cell carcinomas also had bladder stones (calculi). Microscopically, most of the carcinomas had transitional-like cells that formed protrusions into the bladder lumens. Some had papillary areas. The carcinomas had moderate numbers of mitoses and some nuclear pleomorphism. Discrete invasions through the bladder wall occurred in one male rat, but no metastases were evident in the lungs or other tissues.

Kidney:
Chronic inflammation was observed in significantly (P = 0.01) increased incidence in dosed female rats. The dose relationship and intensity of the increased interstitial lymphoplasmocytic infiltrates and cortical fibrosis clearly set these changes apart from the minor inflammatory component that may accompany the progressive nephropathy normally encountered in aging F344/N rats. The changes in the high-dose females were often observed grossly as pitted or roughened renal cortical surfaces. Chronic inflammation of the kidney was not significant in dosed male rats. In those animals in which this lesion was observed, there was no correlation with urinary bladder stones.

Pancreas:
Pancreatic islet-cell carcinomas in male rats occurred with a statistically significant (P =0.034) negative trend (control, 3/44, 7%; low-dose, 0/48; high-dose, 0/45) by the Cochran-Armitage test. The incidences were not significant in pairwise comparisons between the dosed groups and the controls, and these tumors were not observed in female rats. Total pancreatic islet-cell tumors (adenomas or carcinomas) were not significantly different for either sex of rats.

Thyroid:
C-cell carcinoma in the thyroid was observed in female rats with a statistically significant (P = 0.038) positive trend (controls, 0/50; low-dose, 0/49; high-dose, 3/ 50, 6%). Neither the pairwise comparisons of the high-dose group with the controls nor the combination of C-cell adenomas or carcinomas was statistically significant in any of the tests. These tumors were not observed in statistically significant proportions in male rats.

Uterus:
Endometrial stromal polyps were observed in statistically significant (P = 0.017) negative trend (controls, 11/5O, 22 %; low-dose, 7/50, 14 %; high-dose, 2/50, 4 %) and in decreased incidence (P = 0.022) in the high-dose group in a pairwise comparison with the controls. The combined incidence of endometrial stromal polyps and sarcomas was statistically significant (P = 0.017) in the negative direction (controls, 14/50, 28 %; low-dose, 7/50, 14 %; high-dose, 4/50, 8 %). A significantly lower (P = 0.029) incidence in the high-dose group was observed in the pairwise comparisons with the controls. The combined incidence of endometrial stromal polyps and sarcomas in the high-dose group was not significantly different from the historical rate of this tumor in untreated female F344/N rats at the same laboratory (117/759, 15.4 %).

Dose descriptor:

NOAEL

Effect level:

2 250 mg/kg diet

Sex:

male

Basis for effect level:

other: Transitional-cell carcinomas in the urinary bladder.

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

< 4 500 mg/kg diet

Sex:

female

Basis for effect level:

other: Chronic inflammation, kidney.

Remarks on result:

other: Effect type: toxicity (migrated information)

Conclusions:

Under the conditions of this bioassay, melamine was carcinogenic for male rats, causing transitional-cell carcinomas in the urinary bladder. With one exception, urinary bladder stones were observed in male rats that had transitional-cell carcinomas.
Melamine was not carcinogenic for female rats.

Executive summary:

A carcinogenesis bioassay of melamine (>95 % pure) was conducted by feeding diets containing 2250 or 4500 ppm melamine to groups of 50 male F344/N rats for 103 weeks. Groups of 50 female rats were fed diets containing 4500 or 9000 ppm melamine. Groups of 49 male rats and 50 female rats served as controls.

Mean body weights of dosed rats of each sex were lower than those of the controls after week 20. Survival of high-dose male rats was significantly lower (P<0.05) than that of the controls. Survival of all other dosed rat groups was comparable with that of the respective controls.

Transitional-cell carcinomas in the urinary bladder of male rats occurred with a statistically significant positive trend (P = 0.002; controls, 0/45; low-dose, 0/50; high-dose, 8/49, 16 %) and the incidence in the high-dose group was significantly higher (P = 0.016) than that in the controls. A transitional-cell papilloma was observed in the urinary bladder of an additional high-dose male rat. These tumors were not observed in statistically significant proportions in female rats.

Seven of the eight high-dose male rats with the transitional-cell carcinomas also had bladder stones. An association (P = 0.001) was found between bladder stones and bladder tumors in male rats. 

Chronic inflammation, distinguishable from the nephropathy observed in aging F344/N rats, was significantly increased (P = 0.01) in the kidney of dosed female rats (controls, 4/50, 8 %; low-dose, 17/50, 34 %; high-dose, 41/50,82 %) and is attributed to the administration of melamine.

 

The mean body weight of high-dose male mice was lower than that of controls after week 50 of the study. The mean body weights of dosed and control female mice were comparable throughout the study.

Survival of high-dose male mice was significantly less (P < 0.02) than that of the controls. Survival of all other dosed groups was similar to that of the respective controls.

 

Under the conditions of this bioassay, melamine was carcinogenic for male F344/N rats, causing transitional-cell carcinomas in the urinary bladder. With one exception, urinary bladder stones were observed in male rats that had transitional-cell carcinomas.

Melamine was not carcinogenic for female F344/N rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No carcinogenicity studies are available for melamine cyanurate. Neither melamine nor cyanuric acid are classified for carcinogenicity.

Additional information

No experimental data is available regarding the carcinogenic properties of melamine cyanurate itself. Melamine, cyanurate and melamine cyanurate all elicit kidney toxicity by crystal formation. The induction of kidney tumors by melamine crystals at high doses in rats has been discussed in detail in the public literature. Considering the high kidney toxicity and the absence of genotoxic properties further investigations regarding carcinogenicity are not warranted.